Haemolysis and abnormal haemorheology in sickle cell anaemia

Although pulmonary hypertension, leg ulcers, priapism, stroke and glomerulopathy in sickle cell anaemia (SCA) result from the adverse effects of chronic haemolysis on vascular function (haemolytic phenotype), osteoneocrosis, acute chest syndrome and painful vaso‐occlusive crises are caused by abnormal vascular cell adhesion and increased blood viscosity (viscosity‐vaso‐occlusion phenotype). However, this model with two sub‐phenotypes does not take into account the haemorheological dimension. We tested the relationships between the biological parameters reflecting the haemolytic rate (haemolytic component) and red blood cell (RBC) rheological characteristics in 97 adults with SCA. No significant difference in the proportion of patients with low or high haemolytic component in the low and high blood viscosity groups was observed. The RBC elongation index (i.e. deformability) was negatively correlated with the haemolytic component. The RBC aggregates strength (i.e. RBC aggregates robustness) was negatively correlated with RBC elongation index. Sickle RBCs with high density had lower elongation index and higher aggregates strength. In conclusion, (i) the ‘haemolytic’ phenotype is characterized by decreased RBC deformability and increased RBC aggregates strength and (ii) the viscosity‐vaso‐occlusive phenotype is characterized by increased RBC deformability but not always by increased blood viscosity. α‐thalassaemia modulates the haemorheological properties but other factors seem to be involved.     

Soluble CD163 levels in children with sickle cell disease

Sickle cell disease (SCD) is characterized by vasculopathy, which has been causally linked to intravascular haemolysis and high levels of free plasma haemoglobin. Soluble CD163 (sCD163) is implicated in the clearance of free plasma haemoglobin and high plasma concentrations have been linked to arterial disease. We therefore investigated the value of sCD163 as a biomarker in children with SCD, and also measured haptoglobin levels in this population. We measured sCD163 in 25 control children with no haemoglobinopathy, 41 with sickle cell anaemia (HbSS) in the steady state, 27 with HbSS taking hydroxycarbamide, and 7 with HbSC disease. There was no significant difference between sCD163 levels in steady‐state HbSS (1·78 mg/l) and controls (1·81 mg/l) (P = 0·86). However, sCD163 levels were significantly lower in those HbSS children taking hydroxycarbamide (1·35 mg/l) compared to both steady state HbSS (P = 0·004) and controls (P = 0·036). In children on hydroxycarbamide, sCD163 correlated negatively and highly significantly with percentage HbF (R = ?0·76, P < 0·001), and this relationship was absent in those not taking hydroxycarbamide (R = 0·07, P = 0·65). sCD163 is a potentially useful biomarker in children with SCD, and may have a role in monitoring responses to hydroxycarbamide.     

Haemoglobinuria is associated with chronic kidney disease and its progression in patients with sickle cell anaemia

To evaluate the association between haemoglobinuria and chronic kidney disease (CKD) in sickle cell anaemia (SCA), we analysed 356 adult haemoglobin SS or Sβ^o thalassaemia patients from the University of Illinois at Chicago (UIC) and 439 from the multi‐centre Walk‐Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk‐PHaSST) cohort. CKD was classified according to National Kidney Foundation Kidney Disease Outcomes Quality Initiatives guidelines. Haemoglobinuria, defined as positive haem on urine dipstick with absent red blood cells on microscopy, was confirmed by enzyme‐linked immunosorbent assay in a subset of patients. The prevalence of CKD was 58% in the UIC cohort and 54% in the Walk‐PHaSST cohort, and haemoglobinuria was observed in 36% and 20% of the patients, respectively. Pathway analysis in both cohorts indicated an independent association of lactate dehydrogenase with haemoglobinuria and, in turn, independent associations of haemoglobinuria and age with CKD (P < 0·0001). After a median of 32 months of follow‐up in the UIC cohort, haemoglobinuria was associated with progression of CKD [halving of estimated glomerular filtration rate or requirement for dialysis; Hazard ratio (HR) 13·9, 95% confidence interval (CI) 1·7–113·2, P = 0·0012] and increasing albuminuria (HR 3·1, 95% CI: 1·3–7·7; logrank P = 0·0035). In conclusion haemoglobinuria is common in SCA and is associated with CKD, consistent with a role for intravascular haemolysis in the pathogenesis of renal dysfunction in SCA.     

Interplay between coagulation and vascular inflammation in sickle cell disease

Sickle cell disease is the most common inherited haematological disorder that leads to the irreversible damage of multiple organs. Although sickling of red blood cells and vaso‐occlusion are central to the pathophysiology of sickle cell disease, the importance of haemolytic anaemia and vasculopathy has been recently recognized. A hypercoagulable state is another prominent feature of sickle cell disease and is mediated by activation of both intrinsic and extrinsic coagulation pathways. Growing evidence demonstrates that coagulation may not only contribute to the thrombotic complications, but also to vascular inflammation associated with this disease. This article summarizes the role of vascular inflammation and coagulation activation, discusses potential mechanisms responsible for activation of coagulation and reviews recent data demonstrating the crosstalk between coagulation and vascular inflammation in sickle cell disease.     

Moderate endurance exercise in patients with sickle cell anaemia: effects on oxidative stress and endothelial activation

Very few studies have investigated the effects of exercise on the biological parameters involved in vaso‐occlusive events in sickle cell anaemia (SCA). The aim of this study was to test how a mild‐moderate endurance exercise modulates oxidative stress, nitric oxide bioavailability and endothelial activation in SCA patients and healthy individuals. Eleven patients with SCA and 15 healthy subjects completed a 20‐min duration submaximal cycling exercise at ≈45 Watts. Plasma markers of oxidative stress, antioxidant activity, endothelial activation and nitric oxide bioavailability were investigated before and after the exercise. Nitric oxide levels, anti‐oxidant capacity, soluble (s)E‐selectin and sP‐selectin did not change in response to this exercise. Except for the malondialdehyde levels, which increased in the two groups, the other markers of oxidative stress remained unchanged in both groups in response to exercise. Soluble vascular cell adhesion molecule 1 levels were increased at the end of exercise in both groups. sL‐selectin decreased and soluble intercellular adhesion molecule 1 increased with exercise in SCA patients only. The present data suggest that patients with SCA may undertake mild‐moderate physical activities without any acute clinical complications, but care should be taken because oxidative stress and endothelial activation significantly increased in some patients.     

Biomarkers in sickle cell disease

More than 100 different blood and urine biomarkers have been described in sickle cell disease (SCD), with the number increasing rapidly as analytical techniques develop. Nearly all of these biomarkers are abnormal in the steady state, and become more so during complications. The range of abnormalities demonstrates the multisystem nature of SCD and the complex pathophysiology. Some biomarkers indicate damage to specific organs, such as urine albumin:creatinine ratio in nephropathy, whereas others indicate more systemic processes. Biomarkers have been useful in identifying various interrelated pathological mechanisms, including haemolysis, inflammation, hypercoagulability, oxidative stress, reperfusion injury, vasculopathy and endothelial dysfunction. However, most biomarkers correlate closely with other more routine measurements, and also with each other. It is not clear that any provide specific prognostic or clinical information beyond that given by the simple measurement of haemoglobin concentration. The identification of prognostically validated biomarkers in prospective clinical trials would be useful.     

Association between hydroxycarbamide exposure and neurocognitive function in adolescents with sickle cell disease

Patients with sickle cell disease (SCD) are at increased risk for neurocognitive impairments. While disease-modifying treatment, such as hydroxycarbamide (hydroxyurea), may decrease this risk, it has not been systematically investigated in children with SCD. We screened neurocognitive functioning in 103 adolescents with SCD (16–17 years, 50% female) and compared outcomes between patients with a history of exposure to hydroxycarbamide (n = 12 HbSC/HbSβ⁺ thalassaemia; n = 52 HbSS/HbSβ⁰ thalassaemia) and those never treated with hydroxycarbamide (n = 31 HbSC/HbSβ⁺ thalassaemia; n = 8 HbSS/HbSβ⁰ thalassaemia). Demographic distributions were similar between the groups. After adjusting for socioeconomic status, the hydroxycarbamide group had significantly higher scores on nonverbal IQ (HbSC/HbSβ thalassaemia: P = 0·036, effect size [d] = 0·65), reaction speed (HbSS/HbSβ⁰ thalassaemia: P = 0·002, d = 1·70), sustained attention (HbSS/HbSβ⁰ thalassaemia: P = 0·014, d = 1·30), working memory (HbSC/HbSβ⁺ thalassaemia: P = 0·034, d = 0·71) and verbal memory (HbSC/HbSβ⁺ thalassaemia: P = 0·038, d = 0·84) when compared to those who did not receive hydroxycarbamide. In patients with HbSS/HbSβ⁰ thalassaemia, longer treatment duration with hydroxycarbamide was associated with better verbal memory (P = 0·009) and reading (P = 0·002). Markers of hydroxycarbamide effect, including higher fetal haemoglobin (HbF), higher mean corpuscular volume (MCV) and lower white blood cell count (WBC), were associated with better verbal fluency (HbF: P = 0·014, MCV: P = 0·006, WBC: P = 0·047) and reading (MCV: P = 0·021, WBC: P = 0·037). Cognitive impairment may be mitigated by exposure to hydroxycarbamide in adolescents with SCD.     

Serum lactate dehydrogenase activity as a biomarker in children with sickle cell disease

Serum lactate dehydrogenase (LDH) levels were studied in children with HbSS and HbSC in a single institution, and their relationship to cerebral vasculopathy as assessed by transcranial Doppler scanning (TCD). All children with HbSS ( n  = 97) and HbSC ( n  = 18) who underwent a TCD scan in 2006 were studied. LDH levels were higher in HbSS patients than HbSC (581 IU/l vs. 305 IU/l, P  < 0·001). In children with HbSS, LDH correlated significantly with haemoglobin, reticulocytes, aspartate transaminase and creatinine. LDH also correlated positively and significantly with TCD measurements in the middle and anterior cerebral artery circulations in the children with HbSS.     

Association between endothelial dysfunction and otoneurological symptoms in children with sickle cell disease

Objective: To evaluate the association between endothelial dysfunction and otoneurological symptoms and vaso-occlusive phenomena in children with sickle cell disease (SCD).

Methods: Cross-sectional study with 54 children, aged between 6 and19 years of age, of whom 28 had genotype SS and 26 apparently healthy (AA genotype) whose parents or guardians, or the children themselves, filled out a questionnaire designed to assess their otoneurological symptoms. All the individuals were submitted assessment of endothelial function by flow-mediated dilation (FMD) percentage with reactive hyperemia of brachial artery Doppler.

Results: Otoneurological symptoms (tinnitus and/or vertigo) predominated in the SCD group (46.4 vs. 15.4%; p?=?0.006). A negative correlation was observed between FMD percentage and time of evolution of vertigo SCD (r?=??0.432; p?=?0.022) and the linear regression analysis demonstrated that for every reduction in FMD percentage there was an increase in time of evolution of vertigo of 1.79 months (β?=??1.79; p?=?0.022). The positive correlation between episodes of painful crisis and time of evolution of vertigo (r?=?0.3; p?=?0.04).

Discussion: The presence of vascular endothelial damage in the labyrinthine artery in patients with SCD is capable of compromising the semicircular canals, shown by clinical expression of otoneurological symptoms, such as vertigo. In the present study, an association was observed between endothelial dysfunction with otoneurological symptoms and otoneurological symptoms and vaso-occlusive phenomena in SCD.     

Cutaneous microvascular blood flow and reactivity in patients with homozygous sickle cell anaemia

Homozygous sickle cell anaemia (SS disease) involves a high prevalence of skin ulcerations, and background experience concerning the cutaneous microcirculatory flux and reactivity in this disease is very limited. We investigated, by laser-Doppler velocimetry, the microcirculatory cutaneous blood flow and vasoreactivity in 17 patients with SS disease but no cutaneous trophic changes, vs. the corresponding values in 18 normal matched controls. The laser-Doppler probe was placed on the foot dorsum, and recordings were made in the supine and dependent positions, and after post-ischaemic hyperaemia. The venoarteriolar reflex was calculated as the difference between the fluxes in the supine and dependent positions. In both positions, patients with SS disease exhibited clear vasodilation, with larger cutaneous fluxes than those of the controls (P=0.024 and 0.0009, respectively). The venoarteriolar reflex, expressed as a percentage of the resting supine flux, was lower in the patients (P=0.0004). These impairments of the microcirculatory fluxes, which combine a vasodilated state with abnormal vasoreactivity, resemble those observed in patients with chronic venous insufficiency and might be crucial in determining the pathogenesis of the skin ulcerations that occur in SS disease. Laser-Doppler velocimetry seems a suitable non-invasive technique for investigating such cutaneous microangiopathy.     

Association of G6PD202A,376G with lower haemoglobin concentration but not increased haemolysis in patients with sickle cell anaemia

The genetic bases of the highly variable degrees of anaemia and haemolysis in persons with Hb SS are not fully known, but several studies have indicated that G6PD deficiency is not a factor. The G6PD^202A and G6PD^376G alleles and α‐thalassaemia were determined by molecular genetic testing in 261 children and adolescents with Hb SS in a multicentre study. G6PD^202A,376G (G6PD A?) was defined as hemizygosity for both alleles in males and homozygosity in females. Among the participants 41% were receiving hydroxycarbamide. The prevalence of G6PD^202A,376G was 13·6% in males and 3·3% in females with an overall prevalence of 8·7%. G6PD^202A,376G was associated with a 10 g/l decrease in haemoglobin concentration (P = 0·008) but not with increased haemolysis as measured by lactate dehydrogenase, bilirubin, aspartate‐aminotransferase, reticulocyte count or a haemolytic component derived from these markers (P > 0·09). Similar results were found within a sub‐group of children who were not receiving hydroxycarbamide. By comparison, single and double α‐globin deletions were associated with progressively higher haemoglobin concentrations (P = 0·005 for trend), progressively lower values for haemolytic component (P = 0·007), and increased severe pain episodes (P < 0·001). In conclusion, G6PD^202A,376G may be associated with lower haemoglobin concentration in sickle cell anaemia by a mechanism other than increased haemolysis.     

Current developmental screening practices in young children with sickle cell disease

Despite recent developmental screening guidelines, rates of neurodevelopmental disorders (NDDs) remain lower than expected in children with sickle cell disease (SCD). A retrospective chart review identified 276 eligible patients; 214 charts were available for developmental screening and 207 charts for autism-specific screening. Developmental surveillance/screening was conducted in 70% of charts and autism-specific screening in 19% of charts. Validated tools were used in 32% of developmental screenings and 92% of autism-specific screenings. Many children (57%) were screened outside recommended ages. In conclusion, children with SCD are not regularly receiving appropriate developmental screening and surveillance by their healthcare providers.     

Optimal haematocrit and haemoglobin S levels in pregnant women with sickle cell disease

Summary Sickle cell blood has abnormal Theological characteristics and its viscosity is determined by the level of haemoglobin S and the PCV. Rheological studies have shown that alteration of these parameters will improve oxygen delivery to the tissues. However, there is no agreed policy for optimum levels for haemoglobin S and PCV in the management of pregnancies complicated by sickle cell disease. In this study rheological measurements have been performed on pregnant women with sickle cell disease and compared with women without haemoglobinopathy. Potential oxygen delivery was not found to approach normal levels until very low levels of HbS were achieved. The optimal PCV for women with sickle cell disease was found to be 0.26 compared with 0.33 for normal women.     

Daytime steady-state haemoglobin desaturation is a risk factor for overt stroke in children with sickle cell anaemia

Haemoglobin (Hb) desaturation could increase the risk of stroke in sickle cell anaemia (SS) by perturbing endothelial function and limiting oxygen delivery to the brain. We performed a nested case-control study of the Dallas Newborn Cohort to determine whether daytime steady-state Hb desaturation was associated with overt stroke in children with SS. Cases had SS and overt ischaemic strokes. Controls had comparable genotypes but no overt stroke. Cases had lower prestroke steady-state pulse oximetry values (SpO(2)) than controls, and cases' SpO(2) fell even lower as the time to impending stroke decreased. The odds ratio for stroke was 1.32 for each 1% decrease in SpO(2). In conclusion, steady-state Hb desaturation is a risk factor for overt ischaemic stroke in children with SS. Decline in SpO(2) over time further increases this risk. Hb desaturation is easily measured, potentially modifiable, and could be used to identify children with SS at increased risk of stroke.     

Fetal haemoglobin induction in sickle cell disease

Fetal haemoglobin (HbF, α2γ2) induction has long been an area of investigation, as it is known to ameliorate the clinical complications of sickle cell disease (SCD). Progress in identifying novel HbF‐inducing strategies has been stymied by limited understanding of gamma (γ)–globin regulation. Genome‐wide association studies (GWAS) have identified variants in BCL11A and HBS1L‐MYB that are associated with HbF levels. Functional studies have established the roles of BCL11A, MYB, and KLF1 in γ–globin regulation, but this information has not yielded new pharmacological agents. Several drugs are under investigation in clinical trials as HbF‐inducing agents, but hydroxycarbamide remains the only widely used pharmacologic therapy for SCD. Autologous transplant of edited haematopoietic stem cells holds promise as a cure for SCD, either through HbF induction or correction of the causative mutation, but several technical and safety hurdles must be overcome before this therapy can be offered widely, and pharmacological therapies are still needed.     

Genetic determinants of haemolysis in sickle cell anaemia

Haemolytic anaemia is variable among patients with sickle cell anaemia and can be estimated by reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and bilirubin levels. Using principal component analysis of these measurements we computed a haemolytic score that we used as a subphenotype in a genome‐wide association study. We identified in one cohort and replicated in two additional cohorts the association of a single nucleotide polymorphism in NPRL3 (rs7203560; chr16p13·3) (P = 6·04 × 10⁻⁰⁷). This association was validated by targeted genotyping in a fourth independent cohort. The HBA1/HBA2 regulatory elements, hypersensitive sites (HS)‐33, HS‐40 and HS‐48 are located in introns of NPRL3. Rs7203560 was in perfect linkage disequilibrium (LD) with rs9926112 (r² = 1) and in strong LD with rs7197554 (r² = 0·75) and rs13336641 (r² = 0·77); the latter is located between HS‐33 and HS‐40 sites and next to a CTCF binding site. The minor allele for rs7203560 was associated with the −∝^3·7thalassaemia gene deletion. When adjusting for HbF and ∝ thalassaemia, the association of NPRL3 with the haemolytic score was significant (P = 0·00375) and remained significant when examining only cases without gene deletion∝ thalassaemia (P = 0·02463). Perhaps by independently down‐regulating expression of the HBA1/HBA2 genes, variants of the HBA1/HBA2 gene regulatory loci, tagged by rs7203560, reduce haemolysis in sickle cell anaemia.     

Silent infarcts in young children with sickle cell disease

Silent infarcts have been reported most commonly in school-aged children with homozygous sickle cell disease (SCD-SS) and are associated with neurocognitive deficits. However, the prevalence of silent infarcts in younger children with SCD-SS is not well defined. In this retrospective study, brain magnetic resonance imaging and angiography (MRI/A) studies performed before 6 years of age in a cohort of children with SCD-SS were analysed and the prevalence of abnormalities was calculated. Clinical and laboratory parameters were compared between the groups with and without silent infarcts. Sixty-eight of 96 children in the cohort had brain MRI/A performed prior to age 6 years. Of the 65 who were neurologically asymptomatic, 18 (27·7%, 95% CI 17·3–40·2%) had silent infarcts (mean age 3·7 ± 1·1 years, range 1·3–5·9 years). Factors associated with silent infarcts included cerebral vessel stensosis by magnetic resonance angiography, lower rates of vaso-occlusive pain and acute chest syndrome and lower haemoglobin levels. The prevalence of silent infarcts in young children with SCD-SS is similar to that of older children and anaemia and severe vasculopathy may be risk factors.     

Platelet activation in patients with sickle cell disease

Vascular occlusion and vasculopathy underlie much of the morbidity in patients with sickle cell anaemia. Platelets may play a role in this vasculopathy. Samples from 43 patients with sickle cell disease (SCD) were examined for evidence of platelet activation using fluorescent-labelled monoclonal antibodies and flow cytometry. There was increased expression of activation-dependent antigens on the platelets from patients with SCD compared to those from both Caucasian and African-American controls. In addition, SCD patients had increased levels of platelet microparticles. Platelets are activated in patients with sickle cell disease. The contribution of platelet activation to sickle cell pathophysiology is under active investigation in our laboratories.